Project Summary/Abstract The ultimate goal of virtually all immunological research is to generate medicines that would allow precise therapeutic control of the human immune system. Drugs that would control T cell function are sought with the aim of preventing organ transplant rejection and ameliorating autoimmune disease. Unhealthy T cell signaling lies at the root of these clinical problems, and so drugs that would control these diseases would have to do so by controlling T cell signal transduction. Such signaling is mediated by protein-protein interactions (PPI) which (i) can be difficult to study quantitatively, and (ii) can present difficult targets for developing drug therapies. The limiting factor for the latter is that discovery of the rare chemical compounds that are capable of physically affecting PPI is most often accomplished by time- and resource-consuming library screening. We have recently advanced a new technique, IP-FCM, that produces quantitative analyses of the physiologic PPI that mediate T cell signaling with an ease and precision that was previously much more onerous to obtain. The new method is robust, and of high sensitivity, such that both scarce and abundant PPI can be measured. The assay does not require genetic engineering, epitope tagging, or radioactivity, making the method compatible with wild-type mouse, human, or clinical samples. Based on the principles of the technique, we propose to generate a new assay, Multiplex IP-FCM, which will permit the simultaneous analysis of 21+ proteins involved in an extensive network of PPI for T cell signaling (Specific Aim 1). No such technology platform has ever been assembled with this capability. We will prepare this assay for use in the high throughput screening of drug candidates for any effects on the many PPI possible within this protein collection (Specific Aim 2). Thus a single drug screen will be tested for possible effects on many PPI simultaneously, and with the quantitative measurements of the assay, either complete or partial drug activities will be observable. Although our work is focused on T cell signaling, this method will be applicable to any other collections of PPI that may be of interest in other fields of study or classes of disease. It is intended that mounting this new assay will facilitate the discovery of leading compounds that could then be tested for their efficacy as drugs against T cell-mediated diseases.